Reference electrode having a microfluidic flowing liquid junction

ABSTRACT

A flowing junction reference electrode comprises a microfluidic liquid junction member situated between a pressurized reference electrolyte solution and a sample solution. This liquid junction member has an array of nanochannels spanning the member and physically connecting the electrolyte and the sample. While the electrolyte flows through the nanochannels and into the sample, the sample does not substantially enter the nanochannels via diffusion, migration, convection or other mechanisms. The number of nanochannels in the array can be between 10 and 10 8 . Preferably, the nanochannels are substantially straight and parallel to one another. The nanochannels can have widths of between 1 and 500 nanometers, and the width of any one nanochannel is substantially equal to the width of any other nanochannel. The member can be manufactured out a polymer such as polycarbonate and polyimide, and may also be made of silicon, glass, or ceramic.

PRIORITY CLAIM AND RELATED APPLICATIONS

[0001] This application claims priority to, and hereby incorporates byreference herein, U.S. application Ser. No. 09/590,781, filed Jun. 8,2000, issued as U.S. Pat. No. ______ on ______, 2003; U.S. applicationSer. No. 09/738,881, filed Dec. 14, 2000, issued as U.S. Pat. No.______, on ______, 2003; U.S. application Ser. No. 10/361,708, filedFeb. 6, 2003; and U.S. Application Ser. No. 60/138,141, filed Jun. 8,1999.

[0002] This application is a continuation of each of application Ser.Nos. 10/361,708, 09/738,881 and 09/590,781. application Ser. No.10/361,708 is a continuation of application Ser. No. 09/590,781.application Ser. No. 09/738,881 is a continuation-in-part of Ser. No.09/590,781. All of these applications claim priority to Application Ser.No. 60/138,141.

[0003] This invention was made with United States Government supportunder SBIR Phase I and Phase II Grant Nos. DMI-9960665 and DMI-01 10520awarded by the National Science Foundation. The United States Governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

[0004] 1. Field of the Invention

[0005] The invention relates to potentiometric and electrochemicalreference electrodes and, in particular, to liquid junction structuressuch as to be used in electrochemical reference electrodes forelectrochemical measurements of solutions. The invention moreparticularly relates to reference electrodes for use where measurementor control of potential is desired such as with pH or ISE potentiometricsensors used for laboratory analysis, for on-line process monitoring,for field measurements, or in any application where the improvedprecision or extended useful life of the sensor is desirable.

[0006] 2. Description of the Related Art

[0007] The invention is broadly concerned with reference electrodes,such as the reference electrode portion of combination electrodes, andthe reference portion of all potentiometric devices that employ areference electrode to provide the relatively stable reference potentialrequired in various measurements such as electroanalytical measurements,controlled potential coulometry, and polarography, and the like.

[0008] Potentiometric measurements are used widely for the determinationof pH and the detection of other specific ions in a variety of settings,including chemical processes, environmental monitoring, health care andbio-processes. The accuracy of these measurements depends on the abilityto measure the potential difference between a sensing electrode, whosepotential varies with the analyte concentration in the measured samplesolution, and a reference electrode, which ideally would maintain aconstant potential. The physical interface between the referenceelectrode (typically the electrolyte of the reference electrode) and thesample solution is referred to as the liquid junction. The stability ofthe reference electrode, and consequently the accuracy of potentiometricmeasurements, are dependent on the constancy of the liquid junction andmore particularly, the constancy of the potential across the liquidjunction. However, the liquid junction and more particularly, thepotential across the liquid junction are difficult to control andmaintain at a constant level. Typically, it is the change in theliquid-junction potential that introduces error into the electrochemicalmeasurement and results in the need for frequent sensor systemcalibration.

[0009] The errors observed in currently commercially available referenceelectrodes include (1) Transient or kinetic error; such error refers torelatively slow response between measurements, and slow ability to reachequilibrium, typically of five, ten, or fifteen minutes after exposureto extreme solutions. This response is primarily caused by entrapment ofsample solution within the physical junction. Transient errors aretypically a function of the time required to disperse this entrappedlayer of sample solution and obtain a direct interface. The kinetics ofthis error are determined by the duration of prior immersion. The errorsobserved in currently commercially available reference electrodes alsoinclude (2) static error; such error typically refers to persistentoffset after equilibrium is reached. Large static errors are typicallycaused by irreversible entrapment of sample solution deep within thephysical junction structure. The errors observed in currentlycommercially available reference electrodes include (3) stirring error;such error refers to the shift in potential due to or associated withagitation of the sample solution. Stirring error is typically observedwhere there is a rate of agitation or flow of the sample. These errorsexist in potentiometric electrode measurements of sample solutions, buttend to be suppressed in standard buffers where electrode accuracy isbeing checked Therefore, users may see no reason to disbelieve theerroneous readings obtained in non-standard solutions. See D. P.Brezinski, “Kinetic, Static, and Stirring Errors of Liquid JunctionReference Electrodes”, Analyst 108 (1983) 425-442; see also U.S. Pat.No. 4,495,052. These errors are large enough to be of practicalconsequence. These errors often correspond to relatively largedifference in hydrogen ion (H+) concentration or activity. These errors,including those errors described above, tend to bias the measurementsobserved on pH meters by as much as 0.5 pH unit.

[0010] In typical, currently commercially available electroanalyticalmeasurement systems, the interface between the reference electrode'selectrolyte and the sample solution is the liquid junction. The junctionpotential at this sample-reference interface is related to a number offactors; it is an object of every reference electrode design to minimizethe effect of the factors that would cause the liquid junction potentialto drift or to vary in any way over time. Various materials have beenutilized in forming a liquid junction, including porous ceramic rods,porous polymer disks, wood dowls, ground glass sleeves, capillary tubes,agar gels, asbestos fiber bundle, and other porous materials or devices,and the like. These junction structures are, in general, referred to asrestriction devices because their function is to restrict the outwardflow or diffusion of electroyte from the reference electrode. However,one important factor that limits the useful lifetime of a referenceelectrode is that junction structures typically allow the samplesolution to enter the junction structure. This transport of samplesolution into the junction, whether by diffusion, migration, convectionor other mechanism, results in the contamination of the junctionstructure and a resultant undesirable variation in the liquid junctionpotential. Such variation typically necessitates re-calibration of theelectroanalytical measurement system. If this type of contamination ofthe junction continues over time, the junction structure may becomefouled or clogged and develop even larger offset potentials and/orpotentials that chronically drift despite repeated attempts atre-calibration. In addition, sample solution will often transport pastthe junction structure and reach the reference half-cell itself,potentially causing additional adverse reactions.

[0011] Currently commercially available reference electrodes, especiallythose used for potentiometric measurements, are typically constructedbased on one of two distinct designs. Each of these designs is meant toaddress one principle limitation encountered when using referenceelectrodes for making potentiometric measurements. However, each ofthese designs fails to address a distinct principle limitationencountered when using reference electrodes for making potentiometricmeasurements.

[0012] One design category is often referred to as a flowing junctionreference electrode. This design provides a stream of referenceelectrolyte flowing through a porous junction structure or member, in anattempt to provide a relatively uniform liquid junction potential. Whilethis design is typically effective in providing a liquid junctionpotential that is more uniform over time than those of the alternatedesign, flowing junction reference electrodes uniformly require the useof large amounts of electrolyte over relatively short periods of time.Thus, currently commercially available flowing junction referenceelectrodes require frequent maintenance to replenish the supply of thiselectrolyte solution. Furthermore, while flowing junctions are oftendesigned to minimize this use of electrolyte by restricting the flow ofelectrolyte, in such flowing junctions designs the flow velocity isoften reduced to a velocity that is sufficiently low enough so that thesample solution enters the liquid junction structure, typically via masstransport (diffusion, migration, or convection). The presence of thissample solution in the junction structure causes variable junctionpotentials, loss of calibration, clogging of the junction structure,and, over time, failure of the reference electrode. See U.S. Pat. No.5,360,529.

[0013] The alternative design category is referred to as a non-flowing,diffusion junction reference electrode. This design depends on thesubstantially constant diffusion of electrolyte solution through aminimally porous junction structure to provide a steady liquid junctionpotential. While this design is highly susceptible to mass transport ofthe sample stream into the porous structure, the resulting drift inliquid junction potential may be slow enough to be tolerable in certainindustrial applications. While such electrodes require frequentre-calibration, they do not require replenishment of electrolyte to theextent that flowing liquid junction electrodes do. Furthermore, suchelectrodes do not require systems and associated equipment to feed thereference electrolyte to the electrode, as is the case for typicalliquid flowing junction electrodes.

[0014] Both reference electrode designs are in wide use but, based ontheir respective limitations, are typically used in different areas ofapplication. Where precision measurements are more often needed, theflowing liquid junction reference electrode is typically used. Thus theflowing junction design is most commonly used for laboratory referenceelectrodes and clinical analyzers. In the laboratory environment thereference electrolyte may be relatively easily refilled as needed, evenon a relatively frequent basis. Where it is desirable to minimizemaintenance and where precision may be sacrificed to certain degrees,the diffusion junction reference electrode is more often utilized. Thusthe diffusion junction reference electrode is typically used inindustrial potentiometric sensor designs. An industrial sensor that usesa non-flowing, diffusion junction reference will typically requirere-calibration on a more regular basis because of the relatively largeamount of transport of the sample stream into the liquid junctionstructure. It is therefore not unusual for the industrial operator toinstall a new sensor every three months instead of attempting tore-calibrate the old sensor. For this reason, the industrial pH sensorwith a built-in diffusion reference electrode is now a disposable itemin most industrial applications.

[0015] In summary, two principal problems with currently commerciallyavailable reference electrodes are the frequent maintenance requirementof the flowing junction design electrodes and the frequentre-calibration requirements of the diffusion junction design electrodes.More specifically, nearly all flowing junction designs consume largeamounts of electrolyte and this electrolyte needs to be replenished on aregular basis. While there are a few flowing junction designs thatrequire small amounts of electrolyte, these designs have achieved thisby reducing the electrolyte flow to the point that the level oftransport of the sample solution into the liquid junction structurebecomes a limitation. A slow flowing junction reference electrodeperforms little better than a non-flowing, diffusion junction referenceelectrode. On the other hand, the non-flowing, diffusion junctionelectrode requires no electrolyte replenishment but will be subject toslow drift errors due to transport of the sample stream into the liquidjunction structure. This drift typically prevents such referenceelectrodes from being used for precision measurements. Frequently, suchtransport will cause an irreversible instability to develop in thereference electrode that will render it incapable of beingre-calibrated. Because of these inherent shortcomings, sensors employingsuch reference electrodes are often designed to be thrown away andreplaced instead of re-calibrated. As a group, all non-flowing,diffusion junction reference electrodes have a very short operationallife measured in weeks and months and in the best of circumstancesseldom over one to two years

[0016] Accordingly, there is a need in the art for an electrode designthat exhibits both the relatively stable potential of currentlycommercially available flowing junction designs and the relative lack ofthe need to replenish reference electrolyte solution as found incurrently commercially available non-flowing junction designs. Such aneeded design would exhibit a relative stable junction potential overprolonged periods of time, while not exhibiting the various limitationsand drawbacks of currently commercially available flowing junction andon-flowing designs.

SUMMARY OF THE INVENTION

[0017] A microfluidic flowing liquid junction (MLJ) member, for use in avariety of potentiometric devices such as reference electrodes orcombination electrodes, is described. This microfluidic flowing liquidjunction comprises nanochannels in a microfluidic structure that createsa substantially invariant liquid junction potential. The microfluidicflowing liquid junctions comprising nanochannels in a microfluidicstructure also preferably exhibit resistances across the junction memberthat are less than approximately 1 megohm. Low volume of flow throughthe array of nanochannels, and high velocities of electrolyte may beemployed to prevent back diffusion of sample solution into the junctionstructure. Prevention of such back diffusion increases the precision anduseful life of a reference electrode having the described junctionmember. The microfluidic liquid flowing junction member is useful toconstruct highly stable, low maintenance, precision electrochemicalsensors, including reference electrodes.

[0018] A flowing junction reference electrode exhibiting such heretoforeunattainable characteristics is described structurally as comprising amicrofluidic liquid junction member that is situated between a referenceelectrolyte solution and a sample solution. This microfluidic liquidjunction member has an array of nanochannels spanning the member andphysically connecting the reference electrolyte solution and a samplesolution. The reference electrolyte solution flows through the array ofnanochannels and into the sample solution at a linear velocity, and thesample solution does not substantially enter the array of nanochannels.The sample solution does not substantially enter the array via any masstransfer mechanisms such as diffusion, migration, and convection. Asample solution that enters the array at a rate of less thatapproximately 2×10⁻⁹ moles, and preferably less that approximately1×10⁻⁹ moles per day, should be considered as not substantially enteringthe array. The number of nanochannels in the array is preferably betweenapproximately 10⁸ and approximately 10, more preferably less thanapproximately 10⁶, less than approximately 10⁵, and less thanapproximately 10⁴, and most preferably between approximately 10⁴ andapproximately 100. The number of nanochannels may also be, lesspreferably, between approximately 10 and approximately 1000, includingapproximately 10, approximately 40, approximately 100, approximately200, approximately 400, and approximately 800. Also preferably, thenanochannels are substantially straight and are substantially parallelto one another; such an array of nanochannels is herein described asanisotropic. The nanochannels are also preferably coated, and may becoated with, for example, metals, alloys, hydrophilic materials, orhydrophobic materials. The widths of any nanochannels in the array ofnanochannels are preferably substantially uniform, in that the width ofany nanochannel is substantially equal to the width of any othernanochannels in the array. The nanochannels preferably have widths ofgreater than approximately 1 nanometer and less than approximately 500nanometers, more preferably greater than approximately 10 nanometers andless than approximately 100 nanometers, and most preferably 70nanometers. The electrode may be constructed out of any suitablematerial, and is preferably constructed of a polymer, most preferablythe polymer is selected from the group consisting of polycarbonate andpolyimide, and may also be constructed of other structurally strongpolymers, silicon, glass, or ceramic.

[0019] The electrode may also further comprise a pressurized collapsiblebladder, an electro-osmotic pump, or other mechanical pump, or any othermeans for maintaining positive linear flow of the reference electrolytesolution through the array of nanochannels and into the sample solution.The disclosed reference electrode may be used as part of a combinationelectrode along with an appropriate sensing electrode such as a pHelectrode, an ion-selective electrode, a redox electrode, or the like.

[0020] A flowing junction reference electrode exhibiting such heretoforeunattainable characteristics may also be described as comprising areference electrolyte solution flowing through a junction member andinto a sample solution; wherein substantially no sample solution entersinto the junction member via mechanisms of mass transfer such asdiffusion, migration, or convection mechanisms. The linear velocity ofthe reference electrolyte solution flowing into the sample solution ispreferably greater than approximately 0.1 cm per second, more preferablygreater than approximately 0.5, and more preferably greater thanapproximately 1.0 cm per second. The volumetric flow rate of thereference electrolyte solution into the sample solution is less thanapproximately 60 μL per hour, and more preferably less thanapproximately 10 μL per hour. The microfluidic flowing liquid junctionreference electrode is capable of having a lifetime of greater than oneyear, and preferably greater than two, three, four, five, or ten years,during which variations of electrolytic potential are less thanapproximately 1 mV per year, and during which less than approximately100 mL of electrolyte flows into the sample solution, and morepreferably less than approximately 50 mL. The resistance across thejunction member electrode is preferably less than approximately 1megohm.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings, which are incorporated in andconstitute a part of the specification, are included herein toillustrate certain preferred embodiments of the invention and, togetherwith the remainder of the written description and claims providedherein, including the Detailed Description of the Preferred Embodiments,serve to explain the principles of the invention. The accompanyingdrawings are not intended to limit or otherwise define the invention.

[0022]FIG. 1 depicts a schematic cross-sectional view of a referenceelectrode with means for holding the microfluidic flowing liquidjunction in place at the end of the electrolyte reservoir.

[0023]FIG. 2 depicts a detailed schematic cross-sectional view of ameans for holding the microfluidic liquid junction structure in place.

[0024]FIG. 3 depicts a schematic exploded diametric view of the meansfor holding the microfluidic liquid junction structure in place.

[0025]FIG. 4 depicts a schematic cross-sectional view of certainelements of a preferred microfluidic flowing liquid junction structureand a preferred nanochannel array.

[0026]FIG. 5 is an illustrative view representing a single planar,polymer microfluidic flowing liquid junction structure in whichanisotropic nanochannels have been fabricated.

[0027]FIGS. 5 and 6 each depict schematic diametric views illustratingsteps in the fabrication of a multiple planar layer polymer junctionstructure with anisotropic nanochannels and supporting microchannels.

[0028]FIG. 7 depicts a detailed schematic cross-section view showingdetail of the region in which the nanochannels meet a microchannel in apreferred polymer structure.

[0029]FIG. 8 depicts a diametric illustrative view of a microfluidicflowing liquid junction structure having nanochannels and supportingmicrochannels that has been fabricated from one planar element ofsilicone.

[0030]FIG. 9 depicts a schematic cross-section view showing the detailof where the nanochannel meets a microchannel in a silicon microfluidicflowing liquid junction structure.

[0031]FIG. 10 depicts diametric views illustrating steps in thefabrication of a preferred glass microfluidic flowing liquid junctionstructure from multiple planar glass elements.

[0032]FIG. 11 depicts a schematic cross-sectional view of a glassmicrofluidic flowing liquid junction structure.

[0033]FIG. 12 is a plot of the flux (linear flow) through a nanochannelarray and the average velocity (v) through a single nanochannel as afunction of the effective radius of the nanochannel.

[0034]FIG. 13 is a set of concentration profiles in a liquid junction,plotted as a function of velocity, and as described in Equation (7).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0035] A reference electrode is described that comprises a microfluidicflowing liquid junction having a well-defined junction region, saidjunction region containing a reference electrolyte, wherein saidmicrofluidic liquid junction provides a linear rate of flow of saidelectrolyte that is adequate to suppress measurable changes in theelectric potential of the junction for a period of at least one week,and preferably of longer periods including at least one month, at leastthree, six, and nine months, and at least one, one and one-half, two andas long ten years. An electrochemical or potentiometric sensor is alsodescribed comprising a pH electrode, an ion-selective electrode, orredox electrode, and a reference electrode. The reference electrodecomprising means for maintaining a liquid junction potential thatremains stable for a period of at least one week, and preferably forlonger periods including periods of at least one, two, three, six, ornine months, and at least one, one-and-one half, and two and as long asten years.

[0036] By using a novel microfluidic junction structure consisting of anarray of nanochannels, it has been unexpectedly found that heretoforeunattainably stable potentials, low junction potentials, and lowelectrolyte consumption rates for reference electrodes may be produced.These results are preferably attained by using combinations of thenumber of nanochannels and the nanochannel cross-section widths and apositive linear flow velocity for the reference electrolyte through thejunction. The junction structure of the invention may therefore becharacterized by, among other characteristics, (1) high electrolytevelocities to suppress transient, static, and stirring errors; (2)substantially constant junction potentials; (3) substantially constantpotential despite the existence of flow rate and flow velocityfluctuations within the junction; (4) small junction potentials; (5) lowjunction resistance; and/or (6) extremely low consumption ofelectrolyte.

[0037] It is therefore one object of the invention to provide areference electrode with a flowing liquid junction structure that willmaintain a heretofore unavailable relatively constant, invariant, andfixed junction potential, such potential being maintained for extendedperiods of time, including periods of one month to up to one, two,three, and even ten years, without the need to replenish the referenceelectrolyte.

[0038] Another object of the invention is to provide a flowing liquidjunction that functions for relatively prolonged periods of time on arelatively small amount of electrolyte and provides a substantiallyconstant liquid junction potential that is substantially free oftransient errors, static errors, and stirring errors.

[0039] It is another object of the invention to provide a referenceelectrode that will have minimal transient, static, or stirring errorsin sample solutions of extreme pH, solutions having relatively highconcentrations of highly charged ions, and/or solutions having low ionicstrength.

[0040] Another object of the invention is to provide a flowing liquidjunction that is neither a “leak path” nor a “restricted diffusion”junction. In this flowing liquid junction of the invention, there ishydrodynamic transport across the junction structure or member into thesample solution. This hydrodynamic transport is preferably at a velocitysufficiently high to effectively counter back diffusion of the samplesolution into the nanochannels of the junction. Prevention of this backdiffusion contributes to the junction potential remaining stable andfree of transient, static, and stirring errors for prolonged periods oftime of one month to up to one, two, three, and even ten years.

[0041] Another object of the invention is to provide a flowing junctionstructure that provides a constant liquid junction potential over abroad range of electrolyte flow velocities. The liquid junctionstructure provides a constant potential that is relatively andsubstantially free of fluctuations even as the electrolyte velocityvaries within various velocity ranges.

[0042] It is another object of the invention to provide a flowingjunction reference electrode that functions for relatively long periodsof time without the need for replenishment of the reference electrolyteor the associated maintenance. The reference electrode according to theinvention may thus function for times of 1, 10, 20, 30, 40, 50, 60, 70,80, 90 or even 100 years while using less than 100 mL of electrolyte.

[0043] It is another object of the invention to provide a flowingjunction reference electrode that uses such small amounts of electrolytethat the electrode will consume as little as 1 mL of electrolyte peryear. Certain preferred embodiments of the invention allow the referenceelectrode to function for as long as 10, 20, 30, 40, 50, or as long as100 years on only 100 mL of electrolyte, or in other embodiments, lessthan a mL per week, a mL per month, or a mL per six months.

[0044] It is another object of the invention to provide a junctionstructure that consists of an array of nanochannels that provide lowelectrolytic resistance. While each separate nanochannel is high inelectrolytic resistance, the entire array of nanochannels provides ajunction structure having an electrolytic resistance that is relativelylow.

[0045] It is another object of the invention to provide a pressurizedarray of nanochannels that achieves a linear velocity of electrolytenecessary to substantially and effectively counter back diffusion of thesample stream into the junction and thus avoid transient, static, andstirring errors.

[0046] It is another object of the invention to provide a pressurizedarray of nanochannels that achieves a linear velocity of electrolytenecessary to substantially and effectively reduce fouling and blockageby gas bubbles or particulate matter.

[0047] It is another object of the invention to provide a pressuredifferential across the array of nanochannels, through which referenceelectrolyte flows. The volume of each typical nanochannel in the arrayis sufficiently small that high electrolyte velocity can be achieved forprolonged periods of time with the use of extremely small volumes ofelectrolyte. These prolonged periods of time can be as long as years andeven decades.

[0048] It is another object of the invention to provide a referencejunction structure sufficiently robust to function in a processindustrial environment and sufficiently small to be incorporated as abasic building block into portable microfluidic module-based analyticaldevices.

[0049] It is another object of the invention to provide a liquidjunction structure that can be miniaturized for compatibility andintegration into microfluidic devices, such as for example hand-heldanalytic devices for use in remote locations, and portable analyticdevices for use in field stations, battlefield hospitals, emergencystations or the like.

[0050] Another object of the invention is to produce a referencejunction structure with a nanochannel array that may be manufacturedwith planar fabrication techniques so that the reference junctionstructure may be batch produced as an integral component of the variousmicrofluidic structures and devices.

[0051] Another object of the invention is to provide an substantiallyinvariant liquid junction structure that can be fully integrated intomesoscale and microscale microfluidic devices.

[0052] It is another object of this invention to provide a liquidjunction structure that can be miniaturized for compatibility andintegration into microfluidic devices. A further, related object of thisinvention is to provide a liquid junction structure, the manufacture ofwhich may be achieved through the use of current microfabricationtechniques.

[0053] A device need not attain even one of these objectives to bewithin the scope of the invention.

[0054] General Discussion of The Uses and Design of Reference Electrodes

[0055] The microfluidic flowing liquid junctions and referenceelectrodes incorporating such microfluidic flowing liquid junctions, asdisclosed herein, expand the use of electrochemical monitoring to remoteand/or hazardous sites, and to in-line process conditions. Their usewill result in lower cost and improved efficiency of monitoring andcontrolling chemical and biological industrial processes. A referenceelectrode that extends the useful lifetime of a sensor and maintains acalibration for prolonged periods dramatically reduces maintenancerequirements, increases efficiency, and decreases costs.

[0056] Reference electrodes are most typically used for example in thefollowing way: In the measurements of ion concentration of solutions, areference electrode is commonly employed in conjunction with a sensingelectrode, such as a glass pH electrode, with both electrodes immersedin the test solution. The potential difference between the twoelectrodes is a function of the concentration of the specific ion insolution. A typical example is the conventional pH meter and pHelectrode pair used for measuring hydrogen ion concentrations ofsolutions.

[0057] Reference electrodes are also frequently used in conjunction withan ion-sensing electrode such as a pH electrode or a redox electrode,either separately or in combination, to measure the activity (which is afunction of the concentration) of a given ion in a sample solution. Thetwo electrodes, for example, the reference electrode and theion-selective electrode or the reference electrode and the redoxelectrode, both of which are immersed in the sample solution, typicallyare connected to a means of measuring the potential difference betweenthe two electrodes, for example, an electrometer. The referenceelectrode is expected to provide a constant electromotive force orpotential against which the potential of the ion-selective electrode iscompared. The latter potential consists of a constant component from theelectrochemical half-cell of the ion-selective electrode and a variablecomponent which is the potential across the sensing membrane and whichis dependent upon the activity (concentration) of the ion beingmeasured. The variable component, then, is readily correlated with ionactivity (concentration) by known means. To give accurate results, thepotential of the reference electrode should not change with thecomposition of the sample.

[0058] When used in such applications, reference electrodes are meant toestablish a relatively constant or stable potential, which in an idealsituation is independent of the composition of the liquid sample, but inpractice varies with the liquid junction potential. The liquid junctionpotential is the potential difference, created across the interfacebetween the sample solution and the reference electrolyte. Thisinterface is typically present at the junction member. The junctionpotential will vary with varying dilution and varying ion compositionbetween sample and electrolyte. These variations affect the measuredresults and they will become imprecise or misleading over time.

[0059] A reference electrode is typically comprised of an internalhalf-cell supported in a tube containing a salt solution, the tube ofsalt solution being known as a salt bridge. The salt bridge solution isa strong equitransferent salt solution such as potassium chloride orpotassium nitrate. Electrical connection between the salt solution andthe sample or test solution is made by liquid flow through a suitablyformed aperture or passage in a tube, generally referred to as theliquid junction structure or the leak structure. Sometimes the entireunit consisting of the internal half-cell structure, the tube, the saltsolution, and the liquid junction structure is referred to as ahalf-cell; however, for the present specification, the entire unit willbe referred to as a reference electrode.

[0060] Definitions

[0061] As used herein, the term “nanostructures” refers to assembliesthat have dimensions in the range of approximately 1 to approximately500 nm. Accordingly, “nanochannels” refer to channels having widths ofapproximately 1 to approximately 500 nm.

[0062] As used herein, the terms “mass transfer” and “mass transport”each refer to mechanisms for the flow of mass including diffusion,migration, and convection.

[0063] As used herein, the phrase “the sample solution does notsubstantially enter the array of nanochannels” refers to the substantialabsence of back diffusion of the sample solution into the nanochannelsof the junction where such back diffusion would measurably alter thepotential of the reference electrode.

[0064] As used herein, the term “microfluidic” refers to a structure ordevice having channels or chambers which are generally fabricated at themicron or submicron scale. Such structures and devices preferably haveat least one cross-sectional dimension in the range of about 10 nm toabout 500 microns. Techniques commonly associated with the semiconductorelectronics industry, such as photolithography, wet chemical etching,etc, are typically used in the fabrication of microfluidic structures.Such structures may be batch fabricated in, for example, silicon,polymers (including plastics), ceramic, glass, and quartz, using planarintegrated circuit fabrication techniques.

[0065] As used herein, “fluid mechanics” refers to the study of motionand control of fluids. Micromachined fluid components offer thepotential of revolutionizing applications where precise control of fluidflow is a necessity. Microfluidic systems comprising nozzles, pumps,channels, reservoirs, mixers, oscillators, and valves have been used ina variety of applications including drug dispensation, ink-jet printing,and general transport of liquids, gasses, and liquid/gas mixtures. Theadvantages of these devices include lower cost, enhancement ofanalytical performance, and lower consumption of reagents.

[0066] As used herein, the term “half-cell electrode” means thesolid-phase, electron-conducting contact with the half-cell electrolyte,at which contact the half-cell oxidation-reduction reaction occurs whichestablishes the stable potential between the half-cell electrolyte andthe contact. See, e.g., U.S. Pat. No. 4,495,052.

[0067] As used herein, the term “electrochemical” refers to any useand/or sensor that exploits electrochemistry; and includes within it theterm “potentiometric.”

[0068] Manufacture of the Invention

[0069] Microfabrication of electrochemical sensors using integratedcircuit (IC) technology has been challenged by the failure toincorporate a true reference electrode into the structure. See MarkMadou, “Fundamentals of Microfabrication,” 1997, CRC Press, pg. 469.There is great potential for developing simple devices that areinexpensive, easy to fabricate, disposable, and highly sensitive. Thesedevices can prove to be simple miniaturized diagnostic tools for variousstate-of-health indicators.

[0070] Back diffusion of sample solution into the physical junctiongenerates a junction potential that not only shifts the calibration(generating static error) but may also cause the sensor signal to driftat any measurement point (generating transient error). Such backdiffusion greatly increases the frequency of calibration required toobtain precise data from the electrochemical sensor. This increases thecost of ownership and places limits on the amount of time that such adevice can function unattended. This is especially a problem for remotesensing devices that monitor water chemistry in lakes and streams andhave a need to operate for extended periods of time without maintenanceor recalibration.

[0071] Most attempts to minimize back diffusion require a flowingjunction structure that needs large amounts of electrolyte and periodicrefilling of the electrolyte reservoir and other associated maintenance.This adds to the operational complexity of the sensor device andincreases the cost of ownership by requiring scheduled maintenance by atechnician. This is especially a problem with remote environmentalmeasuring devices that are deployed to monitor lake and stream waterchemistry.

[0072] Volumetric flow rate and electrolyte consumption are typicallycompromised one for the other; decreasing one parameter increases theother. As stated above, it is therefore an object of this invention toprovide a reference structure that prevents back diffusion whilesignificantly increasing the linear velocity of the electrolyte flowingthrough the nanochannel array and minimizing volumetric flow rate. Thisvelocity suppresses back diffusion of the sample into the referencestructure and enables the reference electrode to be operated forextended periods of time without the need for recalibration.

[0073] Embodiments of the invention provide a junction structure thatemploys an array of nanochannels in a microfluidic structure to achievea high electrolyte velocity while at the same time utilizing very lowvolumetric flow rates and using only sparingly small amounts ofelectrolyte solution. The microfluidic structure with its array ofnanochannels can operate from 1 to 100 years on 100 mL of electrolyte.Alternatively a single milliliter of electrolyte could enable a small,disposable measurement device to operate with laboratory precision from2 weeks to a year in harsh environments such as battlefield fieldhospitals.

[0074] Embodiments of the present invention substantially mitigate theselong standing problems of reference junction stability and electrolyteconsumption. With the embodiments of the present invention,potentiometric sensors systems can function for extended periods of timewithout the need for recalibration or electrolyte replenishment.

[0075] Embodiments of the present invention provide a microfluidicreference junction structure that enables precise potentiometricmeasurements to be made with devices and systems that operate remotelyand without maintenance for long periods of time.

[0076] This reference structure can be miniaturized for compatibilityand integration into microfluidic devices. Such miniaturization can besubject to performance and stability trade off's with existing junctionstructures. The microfluidic flowing liquid junction described hereinachieves its superior performance because of its nanoscale structure. Itis already small enough to be included as a subcomponent in a microscaledevice such as a disposable microfluidic chip, disk, or block. Yet thesame microfluidic flowing liquid junction structure is robust enough tobe readily utilized as the liquid junction of a macroscale industrialin-line sensor assembly or a mesoscale analytical handheld device.

[0077] A reference electrode with an substantially invariant liquidjunction potential using an innovative combination of microfluidic andnanotechnology is described. The variability of the liquid junctionpotential is a significant factor in the accuracy of potentiometricmeasurements. Removing this variable will result in potentiometricmeasurements with improved stability, precision and reproducibility. Areference electrode with an substantially invariant liquid junction iscapable of sustaining a single calibration for prolonged periods.Reducing the calibration and maintenance will diminish the cost andenhance the ability to monitor remote and hazardous sites.

[0078] The reference electrode described herein preferably usesmicrofluidic concepts to incorporate a nanochannel array for the liquidjunction structure. This microfluidic flowing liquid junction preferablymaintains a constant potential reproducible to ±0.5 mV (˜0.01 pH unit)and preferably has a life in excess of one year. An important factor isthe stability of the liquid junction. In an electroanalytical system theinterface between the reference electrolyte and the sample solutionconstitutes the liquid junction. Unless these two solutions have thesame initial composition, the system will not be at equilibrium. Thoughthe liquid junction region is not at equilibrium, if it has acomposition that is effectively constant, then the reversible transferof charge through the region can be considered. See Bard, A. J.;Faulkner, L. R. Electrochemical Methods; John Wiley & Sons: New York,1980; pp. 61-64. Providing an adequate outward flow of junctionelectrolyte serves to suppress changes in the junction potential. SeeBrezinski, D. P. The Analyst 1983, 108, 425. Maintaining a constantcomposition, and narrow, well-defined liquid junction region, thereforeprotects the reference electrode's liquid-junction potential stability.The system uses small volumes of electrolyte to make it a practicaldevice for operation for one year or more with a reduced level or nomaintenance.

[0079] Factors that affect the liquid junction potential includetemperature, ionic strength, and transport of ionic and molecularspecies across the reference structure. The most stable and reproduciblereference electrodes use a flowing-liquid junction. The continuous flowof electrolyte maintains a constant rate of ion transport across theinterface. In addition, the constant flow of electrolyte also preventsback diffusion of the sample into the reference electrolyte. However, aconventional flowing junction can use large quantities of electrolyteand require substantial maintenance, which is impractical in mostindustrial applications.

[0080] The microfluidic flowing liquid junction may be comprised ofnanochannel arrays in a structure that results from recent developmentsin microfluidic and nanotechnology. This technology makes it possible togenerate sufficient electrolyte flow through the liquid junction toeliminate contamination of the junction structure, yet use only minimalquantities of electrolyte. The microfluidic flowing liquid junctionpreferably maintains a constant potential for an extended duration oftime, and preferably limits the volume of electrolyte to a volume rateof flow of less than 50 mL per year (6 μL per hour). This allows forreference electrodes, and consequently potentiometric or electrochemicalsensors that require neither maintenance nor recalibration for periodsof preferably at least one week, two weeks, one months, six months, orone year.

[0081] The feasibility of using the microfluidic flowing liquidjunction, may be demonstrated by: (i) determining the electrolyticresistance across the nanochannel arrays; (ii) characterizing the flowof electrolyte through nanochannels as a function of applied pressure,nanochannel material, and nanochannel dimension, (iii) determining therequired electrolyte velocity through a nanochannel to eliminate backdiffusion of the sample solution into the reference electrode, and (iv)building a laboratory reference electrode and demonstrate a stablereference potential using a microfluidic flowing liquid junction.

[0082] Furthermore, the microfluidic flowing liquid junction may befurther optimized as follows: (i) optimizing the electrolyte velocity,nanochannel materials and dimensions, (ii) developing appropriatepumping mechanisms and designs.

[0083] The following description of the present invention is dividedinto two sections. The first section is a technical discussion of themicrofluidic flowing liquid junction and its use in a referenceelectrode, including theoretical and conceptual discussions of theliquid junction and its potential, transport through microchannels, andthe utility of nanochannel arrays. The second section lists anddescribes methods to achieve various tasks, including a discussion ofthe tests and experiments used to demonstrate the functionality of amicrofluidic flowing liquid junction for a reference electrode.

[0084] A Reference Electrode Having a Microfluidic Flowing LiquidJunction

[0085] Prototypes with a microfluidic flowing liquid junction areassembled in the following manner. The preferred junction has a modulardesign for easy exchange of different nanochannel arrays. Six electrodesare constructed so that simultaneous measurements can be made. Thenanochannel array is sandwiched between two silicon rubber gaskets (idapproximately 1 mm). The gaskets can be compressed and sealed to theelectrode body. The electrode allows variable internal pressures. Thereference electrolyte is forced to flow by applying a pneumatic pressureon the reference reservoir. The differential pressure is limited to 40psi or to 100 psi. The reference reservoir contains approximately 50 mLof 4.0 M KCl, and uses a Ag/AgCl reference electrode.

[0086] Determination of the Electrolytic Resistance of the NanochannelArray

[0087] The electrolytic resistance of the nanochannel arrays is measuredby AC impedance. A Solartron AC impedance system is available. Thenanochannel array is clamped between the two halves of a U-tubepermeation cell. Both half-cells are filled with 4.0 M KCl. The workingand reference electrodes are placed in one half-cell (on one side of thearray); the counter electrode is placed in the other half-cell (on theother side of the array). The impedance at high frequencies (e.g., 50kHz to 100 kHz) is real and corresponds to the solution resistance. Inthis configuration, the solution resistance has three components; theresistance in one half-cell, the resistance in the second half-cell, andthe resistance of the nanochannel array. The resistances of thehalf-cells are negligibly small relative to the nanochannel arrayresistance. This may be verified by repeating the same experimentwithout the array. If necessary, the measured solution resistance fromthis experiment will be subtracted from the measured resistance when thenanochannel array is in place. The measured resistances may be comparedto calculated values obtained using eq. (3) below.

[0088] Characterizing the Electrolyte Volumetric Flow Rate and LinearVelocity

[0089] The flow rate and velocity of the reference electrolyte throughthe nanochannel arrays are determined as a function of applied pressure,nanochannel dimension, and nanochannel material. The applieddifferential pressure may be varied from 0 (diffusion) to 40 psig. Theflow rate may be measured by placing the junction in 50 mL of ultra-purewater and measuring its transient conductivity. The experimentallydetermined flow rates may be compared to the predicted flow rates,calculated using eq. (2) below. The linear velocity may be calculatedbased on the pore density and dimensions of the nanochannel array.

[0090] The effect of charged nanochannel walls on the transport of thereference electrolyte may also be studied. Chloride ions readily adsorbon gold surfaces, thus, the Au nanochannels may have a net negativecharge. In this situation, the nanochannels are cation permselective.However, if the nanochannels are pretreated with propanethiol they havean inert, neutral coating, and chloride ions do not adsorb. To determinewhat effect charged walls may have on the transport, flow rates throughAu nanotubules with negatively charged and neutral walls may becompared. This comparison provides useful information on the transportmechanism of permselectivity with pressure driven flow throughnano-sized pores.

[0091] Measuring Back Diffusion as a Function of Linear Velocity ofElectrolyte Solution

[0092] Back diffusion as a function of velocity may be measured using acustom-designed pressure cell. Such a cell consists of feed and permeanthalf-cells. The feed half-cell will contain the 4.0 M KCl. The permeanthalf-cell may be a dilute aqueous solution of a strongly absorbing dyemolecule (e.g., Rhodamine B). The back diffusion of the dye from thepermeant into the feed may be measured spectrophotometrically as afunction of applied pressure. The rate of back diffusion may be measuredby following the time-course of the dye appearance into the feed cell.The velocity of solution flow from the feed to the permeant may bemeasured by monitoring the conductivity of the permeant (due totransport of KCI from the feed) as a function of time. In this way, theminimum solution velocity (feed to permeant) required to eliminate backdiffusion of dye (permeant to feed) into the reference electrode chamberwill be determined.

[0093] Comparing Microfluidic Flowing Liquid Junctions to StandardReference Junctions

[0094] A reference electrode having an microfluidic flowing liquidjunction may be compared to traditional reference junctions to determineits relative potential and utility for reference electrodes. A referenceelectrode with a microfluidic flowing liquid junction may be used for pHmeasurements, and its response may be compared with different referenceelectrodes. The overall stability and performance of a referenceelectrode is determined from (i) transient error, (ii) static errors,and (iii) stirring errors.

[0095] First, when an electrode is transferred from one solution toanother, if any of the first solution is retained within the liquidjunction, the measured potential should have a contribution from theoriginal solution. This is referred to as a memory effect, or transienterror. Notwithstanding any permanent contamination, the liquid junctioncan be renewed by the continuous outflow of reference electrolyte.Memory effects, transient errors, may be determined by measuring thetime required to achieve a steady potential response. The response timesof the microfluidic flowing liquid junction may be compared with typicalflowing, and diffusion-style reference junctions.

[0096] Second, stirring the sample solution can change the measured pH.Stirring can effect the potential measurement in at least two ways.Streaming potentials can build-up from convection of the samplesolution. This becomes evident when the ionic concentration of thereference electrolyte differs from the sample, especially in low ionicstrength sample solutions. In addition to streaming potentials, stirredsample solutions can increase contamination of the liquid junction.

[0097] The effect of pressure in the sample solution may be measured upto 40 psig, in or alternatively to 50, 60, 70, 80, 90, and 100 psig. Thepotential dependence of the microfluidic flowing liquid junction ontemperature may then be determined.

[0098] Performance of the Microfluidic Flowing Liquid Junction OverExtended Times

[0099] The microfluidic flowing liquid junction references may be placedin standard pH buffers for extended periods. The long-term testing mayalso be conducted in different media, including wastewater and soils.The microfluidic flowing liquid junction preferably retains itscalibration to within 0.5 mV over a 24-hour period in adverse testconditions. However, a microfluidic flowing liquid junction preferablysustains a single calibration for even greater prolonged periods oftime.

[0100] Certain Preferred Aspects of The Microfluidic Flowing LiquidJunction

[0101] Certain preferred aspects of the invention, many of which arefurther elucidated through the specific examples described herein andmany of which may be observed in the various embodiments of theinvention, are as follows:

[0102] According to a preferred aspect of the invention, there isprovided an array of electrolyte flow channels in the junction member.As shown herein, an array, as opposed to a single channel lowers theoverall junction resistance while minimizing electrolyte consumption.Each channel can be very high in resistance while the sum resistance ofall the channels of an array will be several orders of magnitude lowerin resistance. Without an array, or plurality, of channels the junctionstructure resistance would typically be too high for practical use.

[0103] According to another preferred aspect of the invention, there isprovided an array of nanochannels in the junction member. Channelshaving internal diameters in the lower end of the nanometer range (forexample, less than approximately 100 nm or approximately 70 nm) permitachieving the preferred elevated electrolyte solution linear velocityand the substantially constant liquid junction potential while consumingonly relative small amounts of electrolyte solution. The array ofnanochannels may also comprise approximately 10³, 10⁴, 10⁵, or 10⁷nanochannels. The volume rate of flow is preferably less thanapproximately 50 mL per month, and may also be less than approximately 2liters, 1 liter, 500 ml, 300 ml, 250 ml, 200 ml, 150 ml, or 100 mL permonth, and more preferably less than approximately 50 mL per year, andmay also be less than approximately 2 liters, 1 liter, 500 ml, 300 ml,250 ml, 200 ml, 150 ml, or 100 mL per year. Also, the linear flow rate,dependent on the radii or effective width of the nanochannels employed,is preferably greater than approximately 0.1 cm per second, and,depending on the radii or effective width of the nanochannels, may begreater than 0.0001 cm, 0.001 cm, and 0.01 cm per second.

[0104] According to another preferred aspect of the invention, there areprovided anisotropic channels in the junction member. Such channels aresubstantially straight and parallel to one another, and with uniformpore size provide substantially uniform distribution of flow throughsubstantially all channels. Such channels may preferably be preparedaccording to the “template synthesis” method described herein and inHulteen, J. C.; Martin, C. R. J. Mater. Chem. 1997, 7, 1075.

[0105] According to another preferred aspect of the invention, there areprovided channels having internal diameters of less than approximately100 nanometers or approximately 70, 50, 40, or 30 nanometers. Channelsof these dimensions enable obtaining the preferred combination ofelectrolyte flow velocity, minimum electrolyte consumption, and arrayresistance.

[0106] According to another preferred aspect of the invention, there areprovided channel lengths greater than approximately 100 nanometers andless than approximately ten microns. Channels at this dimension range(or smaller) also enable obtaining the preferred combination ofelectrolyte flow velocity, minimum electrolyte consumption, and arrayresistance.

[0107] According to another preferred aspect of the invention, there isprovided a number of channels less than approximately one-hundredmillion (10⁸). Arrays with fewer than this number of channels enable adesirable combination of electrolyte flow velocity, minimum electrolyteconsumption, and array resistance.

[0108] According to another preferred aspect of the invention, there isprovided a driven flow with high electrolyte velocity greater thanapproximately 0.1 cm/sec. Flow velocity is a factor in determining thepreferred flow rate of electrolyte through the junction. Velocities atthis rate or higher are necessary to substantially prevent penetrationof each nanochannel by sample solution. Contrary to the commonly usedtechnique of restricting the flow rate (volume and velocity) to minimizeelectrolyte consumption, preferred embodiments of the present inventiongreatly accelerate velocity in a nanochannel structure while usingrelatively small amounts of electrolyte.

[0109] According to another preferred aspect of the invention, there isprovided reduced volumetric consumption of electrolyte. Flowing junctiondesigns traditionally use relatively large quantities of electrolyte andneed frequent replenishment and associated maintenance. The designparameters of this reference junction provide superior electrolytevelocity with vastly reduced flow volume of reference electrolyte. Forexample, as little as one mL per year, is consumed under standardoperating conditions. Preferred embodiments of the invention providejunction designs that can function for prolonged periods of time withoutthe need for electrolyte replenishment and minimal contamination of thesample. Certain embodiments of this invention can, for example, operateup to 90 years with only 100 mL of electrolyte.

[0110] According to another preferred aspect of the invention, there isprovided a low junction resistance: having a resistance across junctionof less than approximately 1000 kiloohms (1 megohms). The microfluidicflowing liquid junction electrode is shown to achieve high velocity andlow volume electrolyte use without sacrificing junction resistance.

[0111] According to another preferred aspect of the invention, there isprovided a junction that maintains a stable junction potential over awide range of junction flow rates and flow velocities. Unexpectedly, thenovel junction does not generate a different internal potential atdifferent flow rates or flow velocities. Such a result is contrary toprior teachings. This unexpected property alleviates the need formaintaining a constant flow rate or velocity. Importantly, in apressurized driven device, the flow rate will decrease as theelectrolyte is depleted. Contrary to teachings and expectations, thejunction potential has remained constant over a wide range of pressuresand flow rates. For this reason, the electrolyte solution may be held ina flexible, pressurized collapsible bladder.

[0112] According to another preferred aspect of the invention, there isprovided a reference electrode that may readily be integrated with anyknown variety of sensing electrode to make a combination sensor.

[0113] According to another preferred aspect of the invention, there isprovided a combination sensor that may employ a battery poweredcompensating circuit. The circuit is designed to substantially null theinherent offset in the sensor and maximize the slope of the sensorresponse between two standards.

[0114] According to another aspect of the invention, it becomesunnecessary to maintain a constant pressure across the junction. Thepressure may vary from high as 40 psig to as low as 10 psig and maintainsubstantially no error.

[0115] According to another aspect of the invention, various mechanismsmay be used to maintain desired flow of electrolyte solution through thejunction member. For example, a pneumatic driven flow or pump, such as acollapsible bladder, or electro-osmotic flow or pump orelectro-hydrodynamic flow or pump may also be used. Also, for example, amechanical pump or flow such as a piston-driven pump or flow may beused, or a spring-driven piston pump or flow, or a piezo-electric flowor pump or an electro-hydrodynamic flow or pumps may be used. Such pumpsare well known in the art and are described by Marc Madou in“Fundamentals of Microfabrication”, 1997, CRC Press, pg. 431-433.

[0116] According to another aspect of the invention, the inner walls ofthe microfluidic flowing liquid junction may be physically or chemicallymodified to alter the flow of electrolyte. For example, the inside wallsof the structure may be coated with substances to enhance flow ofelectrolyte. Also, for example, the inside walls of the structure may beplated with metals such as gold, platinum, or palladium or anothernon-reactive metals or alloys or combinations thereof to increasefunctionality and to effect additional functionality or performancegains. Also, for example, the walls may be made hydrophilic by theaddition of for example, a hydrophilic polymer such as a polythiol orpolyvinylpyrolidone (PVP) or a hydrophobic material. Also a surfactantmay be added to the electrolyte to alter the flow of electrolyte throughthe nanochannels, especially of the smaller nanometer structures.

EXAMPLES

[0117] The microfluidic flowing liquid junction and associatedelectrodes of the invention are described in terms of severalembodiments. These embodiments are preferred and comprise microfluidicliquid junction structures with nanochannel arrays fabricated from avariety of specific materials. Each preferred structure may befabricated, according to techniques known in the art, into a thin waferor membrane, preferably round, that can be mounted onto the end of areference electrode structure. Each junction structure permitselectrolyte flow through a nanochannel array from the internalelectrolyte reservoir of the reference electrode into the samplesolution.

[0118]FIG. 1 depicts a representative diagram of an exemplarypotentiometric reference electrode 100 with a microfluidic liquidjunction structure 102 according to the present invention. The referenceelectrode 100 comprises of a chamber 114 that has a seal 120 on one endand a compression means 122 for sealing the junction structure 102 inplace at the other end. The reference electrode 100 includes anelectrochemical half-cell 108, an electrical conductor 118, and areservoir of reference electrolyte solution 110. The electrolytereservoir 110 is contained in a flexible elastomer reservoir bag 112that separates the electrolyte reservoir 110 from the compressed gas 116that fills the rest of the chamber 114. The compressed gas 116compresses the reservoir bag and the electrolyte therein and by thismeans drives the electrolyte 110 through the aperture 130 and into andthrough the microfluidic flowing liquid junction member and out theorifice 132 and into the sample stream (not shown). In this manner thereference electrode 100 shown in FIG. 1 utilizes the microfluidicflowing liquid junction structure 102 to make electrolytic contactbetween the internal electrochemical half-cell 108 and the samplesolution (not shown).

[0119]FIG. 2 depicts a cross-sectional view of the compression means 122that seals the microfluidic liquid junction 102 structure onto the endof the reference electrode chamber 114. The threaded retainer ring 124compresses the microfluidic liquid junction structure 102 against theo-ring 126 and the gasket 128 and thereby seals it into the end of thereference electrode chamber 114. The pressurized electrolyte 110 ispushed through aperture 130 and into and through the microfluidicsliquid junction structure 102 then out of the orifice 132 and into thesample stream (not shown).

[0120]FIG. 3 depicts an exploded diametric view of the compression means122. In this example of the embodiment the microfluidic liquid junctionstructure 102 is a round planar element.

[0121]FIG. 4 depicts a schematic cross-section of the microfluidicliquid junction structure 102 in its most elementary form, a singleplanar element. As shown, the microfluidic liquid junction structure 102is fabricated in a planar substrate 104. Suitable substrate materialsare generally selected based upon cost, ease of fabrication, dimensionalstability, mechanical strength, and compatibility with the conditionspresent in the particular environment that the structure will beoperating in. Such conditions can include extremes of pH, temperature,ionic concentration, and presence of organic solvents. Useful substratematerials include glass, quartz, ceramic, silicone, polysilicone, aswell as polymeric materials such as polycarbonate, polyimide, and otherplastics typically utilized in microfabrication techniques.

[0122] The junction structure 102 includes a multitude of nanochannels106 fabricated through the substrate 104 and generally perpendicular tothe planar axis of the substrate 104. These nanochannels typically havevery small cross-section dimensions, preferably in the range from about1 nm to 500 nm. It is this small, nanometer scale, cross-sectional widthof the nanochannels 106 that gives them their name. For the particularpreferred embodiments, nanochannels 106 that have cross section widthsof about 10 nm to 100 nm and lengths of about 0.5 μm to 200, 300, or asgreat as 500 μm will work most effectively, although deviations fromthese dimensions are within the scope of the invention.

[0123] The multitude of nanochannels 106 present in the microfluidicliquid junction structure 102 are referred to collectively as the array105. The size of the array 105 is characterized by the number ofnanochannels 106 present in the structure 102. The number ofnanochannels can vary from 10 to 100,000,000. More generally, the numberof nanochannels can be selected from any whole number less than 10⁹, andcan be as low as desired, provided that the effects of the plugging ofone or more channel will not substantially and adversely effect theperformance of the MLJ. For these particular embodiments discussedbelow, an array 105 with a number of nanochannels 106 between 1000 and1,000,000 will work most effectively, though deviations from thesenumbers are within the scope of the invention, as noted above.

[0124] The array 105 of nanochannels 106 is a common element in alldepicted embodiments of the invention and the operationalcharacteristics of a particular array may be predicted by specifyingonly three parameters of the array 105: (1) the cross-sectional width ofthe nanochannel 106, (2) the length of the nanochannel 106, (3) and thenumber of nanochannels 106 present in the array 105. Table 1 providesranges expressed in approximate values preferred ranges, for these threeparameters. TABLE 1 Representative Approximate Ranges for NanochannelArray Parameters Individual Nanochannel 106 Cross-sectional Width Range:1 nm to 900 nm Preferable ranges: 10 nm to 500 nm; 40 nm to 100 nm; 70nm Individual Nanochannel 106 Length Range: 0.5 μm to 500 μm Preferableranges: 0.5 μm to 100 μm; 6 to 10 μm Number of Individual Nanochannels106 in Array 105 Range: 10 to 100,000,000 Preferable ranges: 1000 to1,000,000

[0125] Manufacturing of the array 105 of nanochannels 106 and othermicro- and nano-scale elements and features into the substrate 104 maybe carried out by any number of microfabrication techniques that arewell known in the art. For example, photolithographic techniques may beemployed in fabricating glass, quartz, ceramic, silicone, polysilicone,or “plastic” polymeric substrates with methods well known in thesemiconductor manufacturing industries. Photolithographic masking,plasma or wet etching and other semiconductor processing technologiesdefine microscale and nanoscale elements in and through the substrateand on the substrate's surfaces. Alternatively, micromachining methods,such as laser drilling, micromilling, microgrinding, and the like may beemployed. Similarly, for polymeric substrates, such as plastics, wellknown manufacturing techniques may be used. These techniques includecharged particle bombardment and subsequent wet etching of nanoscale andmicroscale channels through polymeric substrates. Additional techniquesinclude injection molding techniques or stamp molding methods wherelarge numbers of substrates may be produced or polymer microcastingtechniques where substrates with microscale and nanoscale features arepolymerized within a microfabricated mold.

[0126] The microfluidic liquid junction structure 102 may be one planarelement or a laminate of multiple planar elements. The planar elementsmay be attached to each other by a variety of means, including thermalbonding, adhesives, or in the case of glass and some plastics, directfusion by heating to the melting point. The additional planar elementsmay constitute all or part of the array structure, or a rigid supportelement for the array structure element, or such additional layers mayinclude other microfluidic components that integrate into themicrofluidic liquid junction structure to provide increased performanceor additional features. Such additional elements might include microscale sensors and sensing elements that measure parameters such aspressure, flow rate, temperature, electrical resistance,oxidation-reduction (redox) potential, conductivity, and pH. Thesesensors could be utilized to provide feedback concerning the performanceof the potentiometric reference electrode 100 and the microfluidicliquid junction structure 102. Such feedback could be utilized bymonitor instrumentation for preventative diagnostics of the referenceelectrode's 100 performance. Such diagnostics might include determiningthe need for recalibration and predicting and signaling the need forservice well before the reference electrode 100 fails in an on-lineindustrial application.

[0127]FIG. 5 depicts an illustrative diametric cross-sectional view ofthe array 105 of a microfluidic liquid junction structure 102 that isfabricated as a single planar polymer element. The planar element has aspecific density (channels/cm²) of etched anisotropic nanochannels 105.In this embodiment of the present invention specific channel densitiesof generally anisotropic nanochannels were fabricated in 10 μm thicksheets of polycarbonate.

[0128] The first step in the fabrication process was to expose 10 μmthick sheets of polycarbonate to charged particles, mostly heavy ions,in a nuclear reactor. These charged particles perforate the polymersheets and leave “sensitized tracks” in the polymer which aresubstantially anisotropic. By controlling the duration of the exposureto the charged particles, the density of tracks per square centimetercan be controlled to a high degree of reproducibility. These tracks weregenerally uniform in width and straight, or anisotropic, and transversethe polymer sheet in a direction generally 90° to the planar axis of thepolymer sheet. The tracks in the polymer substrate were preferablyetched. This enabled the nanochannels to be selectively etched tochannel diameters of 10 nm and larger. The etching process consisted ofimmersing the polycarbonate sheets in a strong alkaline solution of 6 MNaOH with 10% methanol by volume. To obtain sheets with differentchannel cross-sectional widths the etch times were varied from 1 hour to1 minute.

[0129] In a final step in the fabrication process, the polycarbonatesheets were coated by dipping them into a bath of 0.5%polyvinylpyrrolidone (PVP) solution. The PVP coating is hydrophilic andit enhances the “wetability” of the polycarbonate sheets andnanochannels. To obtain polycarbonate sheets with nanochannels ofcross-sectional diameters of less than 10 nm, the inside walls of the 10m nanochannels were uniformly plated with gold until the nanochannelswere reduced to a cross-sectional width of 5 nm. By these fabricationtechniques, polycarbonate planar elements with a range of nanochannelarrays that contained combinations of ultra small nanochannelcross-sectional widths and low nanochannel densities that were notavailable from commercial sources were obtained and then analyzed.

[0130] By design of the nanochannel array 105 density, a flowingmicrofluidic liquid junction (MLJ) structure 102 was fabricated suchthat it had the desire number of flowing nanochannels 106 exposed toaperture 130 on one side of the microfluidic flowing liquid junctionstructure 102, and the corresponding number of flowing nanochannels 106exposed to orifice 132 on the other side of the microfluidic flowingliquid junction structure 102.

[0131]FIG. 6 depicts steps in the fabrication of a flowing microfluidicliquid junction (MLJ) structure 164 from multiple polymer, polyimideplanar elements 160 and 162 that may be thermal bonded together into onestructure. The two polyimide planar elements can be bonded togetherusing various techniques including those of U.S. Pat. No. 5,525,405(Coverdall et al.) and U.S. Pat. No. 5,932,799 (Moles).

[0132] Anisotropic nanochannels may be fabricated into the polymerpolyimide planar element 162 in the same manner as with thepolycarbonate planar element previously described above. The polyimideplanar element 162 is fabricated to have a specific density ofanisotropic nanochannels. The thicker planar element 160 may also befabricated from polyimide into a honeycomb structure containingrelatively larger, micron scale, microchannels 166 with cross-sectionalwidths on the order of 5 μm to 25 μm in this embodiment. This honeycombstructure of the polyimide planar element 160 adds mechanical strengthto the finished microfluidic flowing liquid junction structure 164without unduly impeding the force of the pressurized electrolyte throughthe nanochannels 106. The polyimide planar element can be fabricatedinto a micron scale honeycomb structure by well known photolithographyand wet etch techniques such as those reported in U.S. Pat. No.5,807,406 (Brauker et al.). Due to the relatively regular geometry ofthe resultant structure the resultant number of active flowingnanochannels 106 may be calculated as the number of nanochannels 106that face a microchannel 166.

[0133]FIG. 7 depicts a schematic cross-section of the resultant flowingmicrofluidic liquid junction structure 164 that is made from twopolyimide planar elements, 160 and 162, that have been thermal bondedinto one structural element. On the average, each of the microchannels166 is connected to a small array 168 with approximately the same numberof nanochannels 106. In operation, pressurized electrolyte 110 entersinto an array 169 of microchannels 166 and exits through the manyconnected nanochannels 106. In this way pressurized electrolyte 110flows through an array 169 of many smaller arrays 168 of nanochannels106. This is a useful technique to build up relatively thick planarstructures that do not unduly impede the pressurized flow of electrolyteinto the nanochannels 106.

[0134] In an alternative embodiment of the invention, additional planarelements of the same or different materials can be bonded on top of themicrofluidic flowing liquid junction structure 164 for additionalfeatures and performance such as additional strengthening structures,valves, or sensing elements. Such fabrication techniques are well knownand are reviewed by Marc Madou in “Fundamentals of Microfabrication”,1997, CRC Press. Referring to FIG. 1, it can be seen that thismicrofluidic flowing liquid junction structure 164 can be sealed intothe exemplary reference electrode 100 by compression means 122. Byproper selection of the nanochannel density of planar element 162 andthe microchannel density of planar element 160, a microfluidic flowingliquid junction structure 164 can be fabricated such that it has anmicrochannel array 169 with the desire number of flowing microchannels166 exposed to aperture 130 and the corresponding, connectingnanochannel arrays 168 with the desired number of flowing nanochannels106 exposed to orifice 132.

[0135]FIG. 8 depicts a flowing microfluidic liquid junction (MLJ)structure 170 that can be fabricated from a single planar element ofsilicone by means of anisotropic plasma etching techniques such as thosereported in U.S. Pat. No. 5,501,893 (Laermer et al.). The microfluidicflowing liquid junction structure 170 has micron scale microchannels 176etched in one side of the structure and connecting nanochannels 106etched through the other side of the structure.

[0136]FIG. 9 depicts a schematic cross-section of the silicone flowingmicrofluidic liquid junction (MLJ) structure 170. The flowingmicrofluidic liquid junction structure 170 has an array 179 ofmicrochannels 176 on one side of the structure that connect to an array178 of nanochannels 106 on the other side of the structure. In thisexemplary embodiment the ratio of nanochannels 106 that connect to eachmicrochannel 176 is one to one. Anisotropic plasma etching can fabricatehigh aspect ratio features in silicone with ratios as high as 20:1.Accordingly, in this embodiment the microchannels 176 can be etched 5 μmwide and 75 μm deep from one side of the structure and the nanochannels106 can be etched 100 nm wide and up to 2 μm deep from the other side ofthe microfluidic flowing liquid junction structure 170.

[0137] Again, the nanochannel array 178 density and the microchannelarray 179 density may be selected such that, a microfluidic flowingliquid junction structure 170 may be fabricated such that it has amicrochannel array 179 with the desired number of flowing microchannels176 exposed to aperture 130 and the corresponding, connected nanochannelarray 178 with the desired number of flowing nanochannels 106 exposed toorifice 132. Such a junction may be designed to exhibit certaincharacteristics suitable to any use.

[0138]FIG. 10 depicts steps in the fabrication of a flowing microfluidicliquid junction (MLJ) structure 184 from multiple glass planar elements180 and 182 that can be thermal bonded or fused together into one planarstructure. Planar element 180 is a solid element of glass, such asCorning 0120 glass, that has a single, relatively large channel 186 inthe center. The channel 186 can be several microns to 1 mm in diameterand it can be fabricated with well known microfabrication techniques.The planar element 182 is a glass disk that has at its center an array188 region of nanochannels. This planar element 182 can be made bymethods reported in U.S. Pat. No 5,264,722 (Tonucci et al.) for themanufacture of nanochannel glass rod. Nanochannel glass rod made by thismethod is essentially a fused bundle of anisotropic glass tubes thateach have a cross-sectional width of just a few nanometer to severalhundred nanometers. Furthermore, the nanochannel glass rod, alsofabricated from Corning 0120 glass, can be clad in non-porous glass sothat just the core of the resultant glass rod is made up of an array 188of nanochannels. A single planar cross section 182 of this rod can becut to use as the nanochannel array 188 of the present embodiment of thepresent invention. The width of the nanochannels and the number ofnanochannels can be precisely controlled by the fabrication methodsreported in U.S. Pat. No. 5,264,722 (Tonucci et al.). The length of thenanochannels in the array 188 length can be controlled by cutting across-section of the rod and grinding it to the desired thickness.

[0139] Where both glass planar layers, 180 and 182, are made from thesame glass, they may be fused together into a single flowingmicrofluidic liquid junction (MLJ) structure 184 by scientific glassblowing techniques well known to those skilled in the art.Alternatively, they may be thermally bonded by the techniques disclosedand reviewed by Marc Madou in “Fundamentals of Microfabrication”, 1997,CRC Press.

[0140]FIG. 11 depicts a schematic cross-section view of the glassflowing microfluidic liquid junction (MLJ) structure 184. The flowingmicrofluidic liquid junction structure 184 has a single large channel186 on one side and a corresponding, connecting array 188 ofnanochannels 106 on the other side. The planar element 180 lendsmechanical strength to the planar element 181 in this embodiment of thepresent invention once they are bonded or fused together into the singleplanar flowing microfluidic liquid junction structure 184.

[0141] As before, by design of the nanochannel array 188 density and thesize of the single channel 186, a microfluidic flowing liquid junctionstructure 184 may be fabricated such that the single channel 186 alignswith the aperture 130 and the corresponding, connected nanochannel array188 with the desired number of flowing nanochannels 106 are exposed toorifice 132. TABLE 2 Representative Operational Specifications OfFlowing Microfluidic Liquid Junctions Electrolyte Linear Velocity Range:greater than approximately 0.1 cm/sec Preferable range: greater thanapproximately 1.0 cm/sec Electrolyte Volumetric Flow Rate Range: lessthan approximately 1500 μl/day (500 ml/yr) less than approximately 60μl/hr Preferable range: less than approximately 150 μl/day (50 ml/yr)less than approximately 6 μl/hr Electrical Resistance Range: less thanapproximately 1 megohm Preferable range: less than approximately 100kohm

[0142] Experimental and Theoretical Data Based Upon Experimental Data

[0143] Table 3 and 4, presented below, detail certain actual physicaland potentiometric characteristics, and estimated physical andpotentiometric characteristics based upon and extrapolated from theactual physical and potentiometric characteristics, of microfluidicflowing liquid junctions of the invention having various structuralcharacteristics.

[0144] Table 3 provides experimental test data for reference electrodeshaving exemplary flowing microfluidic liquid junction (MLJ) structureswithin the scope of the present invention. Transient, static andstirring errors were determined in standard pH 7 buffer solutions afterconsecutive exposures to the test solution. The potential was measuredagainst a pH-sensitive glass electrode. The exemplary MLJ structurematerial was obtained from Osmonics Laboratory Products (Westborough,Mass., USA). The Osmonics part number for the 30 nm nanochannel MLJmaterial, P/N KN3CP01300; the Osmonics part number for the 50 nmnanochannel MLJ material, P/N KN5CP01300. The BJC Model 9015, P/NC2451C-12A, with typical commercially available diffusion junctionreference electrode was obtained from Broadley-James Corp. (Irvine,Calif., USA)

[0145] Table 4 provides the estimated resistance, velocity and lifetimeof exemplary MLJ structures within the scope of the present invention.Table 4 was generated based on the actual, experimentally determineddata derived from a MLJ structure with 1,000 10-μm long, nanochannelshaving widths of approximately 70 nm see bottom row), specially preparedas described herein. TABLE 3 Microfluidic Flowing Liquid JunctionReference Electrode Tests Comparative Reference Electrode Tests:Microfluidic Flowing Liquid Junctions vs. Conventional Non-FlowingDiffusion Junction Test Reference Channel Channel Array Flow RateVelocity Transient Error Static Error Stirring Error Solution ElectrodeWidth Length Size Pressure (μL/hr) (cm/s) (mV) (mV) (mV) pH 4 Buffer MLJDesign 50 nm 6 μm 1,000,000 40 psig 1910 6.4 0.2 <0.1 −0.2 0.1 <0.1 <0.1MLJ Design 30 nm 6 μm 1,000,000 40 psig 70 0.7 0.3 <0.1 <0.1 <0.1 <0.1<0.1 MLJ Design 50 nm 6 μm 1,000,000 10 psig — — 0.1 <0.1 0.1 <0.1 <0.1<0.1 MLJ Design 30 nm 6 μm 1,000,000 10 psig — — 0.2 <0.1 0.1 <0.1 <0.1<0.1 MLJ Design 70 nm 10 μm  1000 40 psig 1.8 13 0.1 0.2 0.1 <0.1 0.30.4 BJC Model 9015 gel electrolyte with non- N/A N/A N/A 1.5 0.6 −2.6−2.9 1.3 0.9 flowing diffusion junction 0.1 M HCl MLJ Design 50 nm 6 μm1,000,000 40 psig 741 2.5 <0.1 <0.1 <0.1 <0.1 0.1 0.2 MLJ Design 30 nm 6μm 1,000,000 40 psig 193 1.8 <0.1 <0.1 <0.1 <0.1 0.2 0.3 MLJ Design 50nm 6 μm 1,000,000 10 psig 114 0.4 <0.1 <0.1 <0.1 <0.1 <0.1 0.1 MLJDesign 30 nm 6 μm 1,000,000 10 psig 20.3 0.2 <0.1 <0.1 <0.l <0.1 0.1 0.1BJC Model 9015 gel electrolyte with non- N/A N/A N/A 5.2 −4.1 −2.3 2.20.6 1.7 flowing diffusion junction 0.1 mM HCl MLJ Design 50 nm 6 μm1,000,000 40 psig 583 2.0 <0.1 <0.1 <0.1 0.2 <0.1 0.2 MLJ Design 30 nm 6μm 1,000,000 40 psig 20 0.19 <0.1 0.2 <0.1 0.3 0.9 1.2 MLJ Design 50 nm6 μm 1,000,000 10 psig 95 0.32 0.1 0.3 <0.1 <0.1 0.1 0.4 MLJ Design 30nm 6 μm 1,000,000 10 psig 47 0.44 0.1 0.2 <0.1 <0.1 0.1 0.2 BJC Model9015 gel electrolyte with non- N/A N/A N/A −3.2 22.5 −2.7 1.4 10.5 12flowing diffusion junction 1 M Tris Buffer MLJ Design 50 nm 6 μm1,000,000 10 psig 374 1.3 −0.2 −0.2 1.4 −0.9 −0.2 0.1 MLJ Design 30 nm 6μm 1,000,000 10 psig 39 0.4 0.9 1.1 −0.2 1.8 0.3 0.2 BJC Model 9015 gelelectrolyte with non- N/A N/A N/A 24 26 −4.5 −1.9 1.6 0.8 flowingdiffusion junction

[0146] TABLE 4 Electrode Characteristics/Lifetime Estimates for VariousJunction Designs Electrode Lifetime Estimates for Selected MLJ Designs(Derived from Junction Linear Flow and Resistance Data) Estimated TotalEst. Linear Est. Lifetime Channel Dimensions Array Size ResistanceVelocity (yrs) Length (# of (k) (cm/s) for 50 mL of ID (nm) (μm)Channels) (kiloohms (40 psig) Electrolyte 10 6 1,000 1,910.83 0.444568.11 10 6 10,000 191.08 0.44 456.81 10 6 100,000 19.11 0.44 45.68 106 1,000,000 1.91 0.44 45.7 30 6 1,000 212.31 3.98 56.40 30 6 10,00021.23 3.98 5.64 30 6 100,000 2.12 3.98 0.56 30 6 1,000,000 0.21 3.980.06 50 6 1,000 76.43 11.05 7.31 50 6 10,000 7.64 11.05 0.73 50 6100,000 0.76 11.05 0.07 50 6 1,000,000 0.08 11.05 0.01 70 10 1,000 64.9913.00 3.17

[0147] Technical Computational and Theoretical Analyses

[0148] Although the invention is not limited to any specific explanationof theory to explain why or under what conditions it performs asdescribed herein, the following technical, computational and theoreticalanalyses are advanced to explain the invention.

[0149] The technical aspects of the microfluidic flowing liquid junctionof the invention are addressed. The theoretical and practicalrequirements of a stable liquid junction are described, and theadvantages of using a microfluidic flowing liquid junction are describedand presented. Calculations and references demonstrate that theinventive use of microfluidic and nanopore technology lead to a stableliquid junction potential.

[0150] Potentiometric measurements are necessarily made using twoelectrodes. One electrode is the sensing electrode, which changes itspotential with the concentration, or activity, of the analyte, e.g.,a_(i) in eq. (1). The other electrode is the reference electrode, whichideally generates a constant half-cell potential, E_(ref), eq. (1). Thepotential at each electrode is characteristic of the physicochemicalstate of the electrode system, for example, the potential depends ontemperature, pressure and the chemical composition of the system. Thepotential of the reference half-cell remains constant by placing theelectrode in a separate compartment with its own electrolyte. Thereference compartment has a conductive path to the sample solution. Thearrangement of the electrode, the reference electrolyte and theconductive path is known as the reference electrode. See Midgley, K.;Torrance, K. Potentiometric Water Analysis, 2^(nd) ed.; John Wiley &Sons: New York, 1991; p 12. The interface between the referenceelectrode and the sample solution is the liquid junction, whichcontributes a potential, E_(junc). The sum of the sensing and referenceelectrode potentials, and the liquid junction potential is the measuredcell potential, E_(cell), eq. (1). $\begin{matrix}{E_{cell} = {\left( {E_{i}^{o} + {\frac{RT}{n\quad F}\log \quad a_{i}}} \right) + E_{{ref}.} + E_{{junc}.}}} & (1)\end{matrix}$

[0151] In order to determine the liquid junction potential accurately(see Bates, R. G. The Determination of pH; John Wiley and Sons: NewYork, 1973), or to minimize it (see Horvai, G.; Bates, R. G. Anal. Lett.1989, 22, 1293), the overall composition of the sample must be known apriori. However, in most chemical analyses the desire is typically notto precisely determine or even minimize the liquid junction potential,but rather that the potential remain substantially constant andunchanging so that a reliable calibration can be made. There istypically no need to determine the liquid junction potential, but thereis a need that the potential be substantially invariant from one testmeasurement to another at a given temperature and pressure. See IUPAC,Quantities, Units and Symbols in Physical Chemistry; Mills, I. Ed.;Blackwell: Oxford, 1993; p 62. Accurate potentiometric measurements thusdepend on the constancy of the liquid junction potential. However, thereis a fundamental limitation with the accuracy in potentiometricmeasurements due to a number of theoretical and practical limitationsincluding a drifting, non-constant liquid junction potential.

[0152] The performance of a reference electrode not only depends on thechemical properties of the electrode, but also on the physicalarrangement of the liquid junction. The four main physical criterion ofsubstantially invariant liquid junction include, see Midgley, D.;Torrance, K. Potentiometric Water Analysis, 2^(nd) ed.; John Wiley andSons: New York, 1991; p 46, (i) the junction structure should beconstant, (ii) stirring or streaming of the sample solution should notaffect the reference potential, (iii) particulate matter from the sampleshould not clog the junction, and (iv) solution from one sample shouldnot be retained in the junction and carried over to the next sample. Theaccuracy of any potentiometric measurement thus depends on the abilityof the liquid junction design to meet these requirements.

[0153] Currently commercially available reference electrodes use anassortment of liquid junction structures and designs to protect thereference electrolyte from the sample. These materials include porousceramic, porous Teflon, wood, asbestos, and various fibers. Designs withdouble junctions, glass-sleeves, and fused salts are also used. Allthese materials and designs are meant to keep the reference environmentconstant. However, even if the reference solution remains unchanged, theliquid junction can become contaminated with the sample solution. Thisinevitably alters the potential of the liquid junction, and requires theelectrochemical sensor to be recalibrated. A changing liquid junction istypically why an electrochemical sensor requires frequent recalibration.

[0154] The most stable, reproducible, and reliable reference electrodedesigns incorporate a flowing-liquid junction. See Covington, A. K.;Whalley, P. D.; Davison, W. Anal. Chim. Acta 1985, 169, 221;Illingworth, J. A. Biochem. J. 1981, 195, 259; Wu, Y. C.; Feng, D.;Koch, W. F. J. Solution Chem. 1989, 18, 641; Ito, S.; Kobayashi, F.; IBaba, K.; Asano, Y.; Wada, H. Talanta 1996, 43, 135; Peters, G. Anal.Chem. 1997, 69, 2362; Lvov, S. N.; Zhou, X. Y.; Macdonald, D. D. JElectroanal. Chem. 1999, 463, 146; Brezinski, D. P. The Analyst 1983,108, 425. The constant flow of reference electrolyte through the liquidjunction helps it maintain a constant composition by the continualrenewal of fresh electrolyte. The disadvantage of using such anelectrode is that it requires considerable maintenance because thereference cell must be frequently refilled with electrolyte. For thisreason, flowing junctions are usually only suitable for the laboratoryenvironment. Another problem of a typical flowing-reference electrode isthat if the sample is at a pressure higher than the reference reservoir,the reference cell will readily become contaminated with the sample.Because of these disadvantages, in recent years, the convenience and lowmaintenance of diffusion-style junctions has replaced the flowing-liquidjunction in industrial application.

[0155] A superior flowing-liquid junction has been developed bycombining microfluidic materials and nanomaterials. The electrolyte hasa continual flow of small, manageable volumes of electrolyte through thejunction with a linear velocity sufficient to eliminate contamination ofthe junction and/or contamination of the reference electrolyte. Themicrofluidic flowing liquid junction provides the superior stability andperformance of a flowing liquid junction yet remain maintenance-free forextended periods of time, including a week, two weeks, a month, sixmonths, a year, or two years.

[0156] When miniaturizing chemical and physical processes, as inmicrofluidics, scaling laws must be considered. In addition, modelingfluid mechanics requires that correct assumptions as to the type of flowbe made. Microfluidics typically have very low Reynolds numbers, Re<1,see Madou, M. Fundamentals of Microfabrication; CRC Press: New York,1997; p 429. where viscous forces dominate. A consequence of viscousflow is that each microscopic fluid element follows a fixed path orstreamline. Any subsequent fluid element, starting at the same point,will follow the same streamline along its entire course. See Giddings,J. C. Unified Separation Science; John Wiley and Sons: New York, 1991;pp. 58-63. Such a flow pattern creates a reproducible, non-varying, andpredictable structure, like that desired in a flowing-liquid junction.To characterize the flow through a liquid junction the velocity profilesmust be determined.

[0157] To determine the velocity profile through a microchannel ornanochannel, all of the external forces acting on the fluid are to bebalanced. First, the Newtonian acceleration (or inertial) forces aresignificant for only a brief moment before steady flow is achieved invery small channels, see Giddings, J. C. Unified Separation Science;John Wiley and Sons: New York, 1991; pp. 58-63, and can be neglected.Second, all of the fluidic elements under consideration terminate as asudden expansion. This implies that the kinetic energy of the fluid isnot transferred from one element to the next. See Gravesen, P.;Branebjerg, J.; Jensen, O. S. J. Micromech. Microeng. 1993, 3, 168.Third, in very small channels gravitational forces may be neglectedsince the pressure required to induce steady flow is typically muchlarger than the gravitational force, i.e., Δp>>pgh. See Giddings, J. C.Unified Separation Science; John Wiley and Sons: New York, 1991; pp.58-63. By neglecting acceleration, kinetic, and gravitational forces weneed only balance the pressure acting against the viscous forces inorder to determine the velocity profile through a microchannel. Flowthrough very small channels is described by the Hagen-Poiseuilleequation, eq. (2). The flux, Q (L/s), or the rate of flow through across-sectional area of a channel is a function of the channeldimensions, the differential pressure, and the properties of thesolution. $\begin{matrix}{Q = \frac{\pi \quad \Delta \quad p\quad r_{o}^{4}}{8\quad L\quad \eta}} & (2)\end{matrix}$

[0158] In eq. (2) Δp is the pressure differential at the two ends of thechannel, r_(o) and L are the radius and length of the channel,respectively, and η is the solution viscosity. (All of the calculationsin this proposal have assumed that the viscosity of the electrolyte isequal to 1.0 cp.) See All pure aqueous KCl solutions have a viscositybetween 0.9 and 1.1 cp. Hai-lang, Z.; Shi-Jun, H. J. Chem. Eng. Data1996, 41, 516. Examination of eq. (2) indicates that Q ∝r_(o) ⁴, thus,simply constricting the cross section of a channel will greatly diminishthe flow through it. However, decreasing the cross-sectional area of achannel increases the electrolytic resistance. The conductance through acylindrical channel can be calculated by using eq. (3). $\begin{matrix}{G = {\frac{1}{R} = \frac{\lambda \quad C\quad \pi \quad r_{o}^{2}}{L}}} & (3)\end{matrix}$

[0159] The electrolytic resistance of the channel is taken as thereciprocal of the cell conductance, G. λ is the electrolyte conductance,C is the electrolyte concentration, A and L are the cross-sectional areaand length of the channel, respectively. λ for a 4.0 M KCl solution is˜10⁻² m² S mol⁻¹. See Handbook of Chemistry and Physics, 71^(st) ed.;Lide, D. R., Ed.; CRC Press: Ann Arbor, 1990. To minimize theelectrolyte flow-rate and the resistance by simply reducing the size ofa single channel is impractical, since the electrolytic resistancerapidly becomes too high when the channel radius <˜1 μm. For example,the calculated electrolytic resistance, using eq. (3), of a 1-mm longchannel with a 1-μm radius containing 4.0 M KCl is ˜8 MΩ. Thisresistance is too high for any realistic consideration, sinceelectrolytic resistance greater than approximately 500 kΩ is outside thecapabilities of typical commercial instrumentation. Fortunately, flowdecreases as the fourth power of the radius while resistance increasesas the square of the radius. Decreasing channel cross section butincreasing the number of channels is a practical way to reduce theelectrolytic resistance while maintaining the desired low flow.

[0160] Preferred embodiments of the present invention use an array ofnanochannels as a liquid junction structure to minimize both the flowrate and electrolytic resistance. For example, while a singlenanochannel with a 5-nm radius, and 6-μm long (see Nishizawa, M,; Menon,V. P.; Martin, C. R. Science 1995, 268, 700) has an electrolyticresistance of approximately 1000 MΩ in 4.0 M KCl (eq. (3)), and forexample, an array of 10⁵ nanochannels will have a resistance <100 kΩ.

[0161] Calculations thus far show that a microfluidic flowing liquidjunction can provide the desired flow control and electrolyticconductivity to achieve a commercial product. Next, the electrolytevelocity needed to minimize the back diffusion of a sample into theliquid junction is calculated. The average solution velocity through asingle nanochannel can be calculated by dividing the flux, Q, eq. (2),by the cross-sectional area of the nanochannel. $\begin{matrix}{v = \frac{Q}{\pi \quad r_{o}^{2}}} & (4)\end{matrix}$

[0162] Using eqs. (2) and (4), the flux through a nanochannel array andthe average velocity (v) through a single nanochannel are plotted inFIG. 1 as a function of the nanochannel radius. The calculations assumea steady pressure difference of 40 psi. The flux is plotted as thesensor life assuming a 50-ml reservoir of electrolyte. The arraycontains 105 nanochannels and is 6 μm long. A 50 mL reservoir will besufficient for continuous operation of a year or more for nanochannelradii less than approximately 30 nm. The radii of the nanochannels ormicrotubes may also have radii of less than approximately 20 nm, lessthan approximately 40 nm, less than approximately 50 nm, or less thanapproximately 60 nm. By increasing the volume of the reservoir, or bydecreasing the number or density of the nanochannels, the lifetime of asensor can be adjusted as needed, as will be appreciated by those ofordinary skill in the art.

[0163] An order of magnitude estimate of the electrolyte velocity neededto diminish diffusion of the sample into the liquid junction iscalculated. A hydrodynamic model is used to model theconvective-diffusion transport through a nanochannel. This modelneglects electrostatic interactions and migrational effects. Diffusionof the sample into the liquid junction is described by Fick's first law,N_(D)=−D∇C, and the convective flux is N_(v)=Cv. The sum of thediffusional and convective fluxes is the total flux, eq. (5).$\begin{matrix}{N = {{{- D}\frac{C}{x}} + {C\quad v}}} & (5)\end{matrix}$

[0164] In eq. (5) C is the concentration of the sample at position x inthe channel. v is the convective velocity of the sample, and isapproximated as the average solution velocity through the channel.Integration of the continuity equation, ∇·N=0, with boundary conditions,C=C_(o) at x=1 and C=0 at x=0, where C_(o) is the initial concentrationof the sample, and I is the length of the nanochannel, yields theconcentration profile for convective-diffusion through a nanochannel.$\begin{matrix}{\frac{C}{C_{o}} = \frac{{\exp \left( \frac{v\quad x}{D} \right)} - 1}{{\exp \left( \frac{v\quad l}{D} \right)} - 1}} & (6)\end{matrix}$

[0165] The concentration profiles in the liquid junction are plotted asa function of velocity in FIG. 13, using eq. (7). Apparently, solutionvelocities >˜0.1 cm/s should be sufficient to exclude the sample fromdiffusing into the reference reservoir. FIG. 12 shows a solutionvelocity of ˜0.1 cm/s can be generated for nanochannel radii >˜10 nmwith a differential pressure of 40 psi. According to these calculations,nanochannels with radii between 10 to 40 nm will yield a constant,non-varying liquid junction that is low in resistance, and is operativefor at least one year.

[0166] Nanochannel arrays thus are shown theoretically to provide theideal approach to solving the liquid junction problem.

[0167] Preferred Laboratory System Embodying the Invention

[0168] A system according to a preferred embodiment of the invention wasassembled. This system was used to test electrodes at controlledtemperatures, pressures and agitation rates. The system consists of a 50mL pressure cell, which can handle pressures as high as 45 psig asequipped. The laboratory test system mimics the different, sometimesharsh environments to which sensors may routinely be exposed inindustrial or field applications. The cell is exposed to temperaturesfor example, within 0.1° C., in a precision temperature bath. Amechanical stirrer provides adequate aeration and mixing of the testsolution. All of the instrumentation is linked to a computer for dataacquisition and archiving of the experimental measurements.

[0169] Theoretical Aspects of the Preparation and Characterization ofthe Nanochannel Array

[0170] The Au nanochannel arrays that were used as the liquid junctionstructure were prepared via a general approach for preparingnanomaterials called “template synthesis.” See Hulteen, J. C.; Martin,C. R. J. Mater. Chem. 1997, 7, 1075. The template method entails thesynthesis of a desired material within the channels of a microporousmembrane. The membranes employed have cylindrical channels withmonodisperse diameters that run the complete thickness of the membrane.Corresponding cylindrical nanostructures of the desired material areobtained within the channels.

[0171] A commercially available microporous polycarbonate filtrationmembrane may be used as the template to prepare the nanochannel arrays.This membrane contains monodisperse and cylindrical pores. An electronicplating procedure is used to deposit Au nanochannels within these pores.See Nishizawa, M,; Menon, V. P.; Martin, C. R. Science 1995, 268, 700;Hulteen, J. C.; Martin, C. R. J. Mater. Chem. 1997, 7, 1075; Hulteen, J.C.; Martin, C. R. J. Am. Chem. Soc. 1998, 26, 6603; Menon, V. P.;Martin, C. R. Anal. Chem. 1995, 67, 1920. This Au plating procedure iswell known in the art.

[0172] The template membrane may be first rinsed in methanol and thenimmersed in a 0.025 M SnCl₂ and 0.07 M in trifluoroacetic acid solution.This results in “sensitization” of the membrane, typically meaning theadsorption of Sn(II) to the channel walls and membrane surfaces. Thesensitized membrane is then immersed into an aqueous solution ofammoniacal AgNO₃. This causes the following surface redox reaction,

2 Ag⁺+Sn(II)→2 Ag^(o)+Sn(IV)  (7)

[0173] and the channel walls and membrane phases become coated withnanoscopic Ag particles.

[0174] These particles act as the initial catalyst for electroless Audeposition. Finally, the membrane may be placed in a gold plating bath,which contains 0.5 mL of a commercially-available gold plating solution,0.127 M Na₂SO₃, 0.625 M formaldehyde and 0.025 M NaHCO₃. The solutionmay be adjusted to pH 10 by dropwise addition of 0.5 M H₂SO₄. Thetemperature of this plating bath is typically maintained at 5° C. Theinside diameter of the Au nanochannels deposited within the pores of thearray is adjusted by varying the plating time, which typically refers tothe immersion time in the Au plating bath.

[0175] This procedure is optionally used to prepare arrays containing Aunanochannels with inside diameters of molecular dimensions (<1 nm). SeeNishizawa, M,; Menon, V. P.; Martin, C. R. Science 1995, 268, 700;Hulteen, J. C.; Martin, C. R. J. Mater. Chem. 1997, 7, 1075; Hulteen, J.C.; Martin, C. R. J. Am. Chem. Soc. 1998, 26, 6603; Menon, V. P.;Martin, C. R. Anal. Chem. 1995, 67, 1920; Petzny, W. J.; Quinn, J. A.Science 1969, 166, 751. Ion-transport in these arrays has been studied,see Nishizawa, M,; Menon, V. P.; Martin, C. R. Science 1995, 268, 700.The resulting nanochannels are ion permselective and may be reversiblyswitched between anion-transporting and cation-transporting states.

[0176] The inside diameters of the Au nanochannels may be readilyapproximated by measuring the flux of H₂ gas across the nanochannelarray. See Petzny, W. J.; Quinn, J. A. Science 1969, 166, 751. See alsoLiu, C., Texas A&M University, College Station; 1991. The nanochannelsamples are then placed in a vacuum oven for at least 12 hours prior tomaking the flux measurements, to remove traces of water or othervolatile species absorbed in the nanochannels. Reproducible values offlux are best obtained when nanochannels are pretreated in this manner.The nanochannel array may then be placed in the gas-permeation cell, andthe upper and lower half-cells evacuated. The upper half is pressurizedto 20 psig with H₂, and the pressure-time transient associated withleakage of H₂ through the nanochannels into the lower half-cell wasmeasured. This is converted to the flux of gas, from which the averagenanochannel diameter may be approximated. Assuming gas-transport througha nanochannel array occurs via Knudsen diffusion, the flux of gas,Q_(gas) (moles cm⁻² s⁻¹), is related to the pore density, n (porescm⁻²), the pore diameter, d (cm), and the membrane thickness, L (cm)using eq. (8). $\begin{matrix}{{({gas})\quad Q} = \frac{8\pi \quad n\quad d^{3}\Delta \quad p}{3\quad M\quad {RTL}}} & (8)\end{matrix}$

[0177] Δp is the pressure difference across the membrane (dynes cm⁻²), Mis the molecular weight of the gas, R is the gas constant (erg K⁻¹mol⁻¹), and T is the temperature (K). In our experiment, we know all ofthe parameters in eq. (8), except d.

[0178] A variety of nanochannel arrays of various sizes and materialsmay be constructed and used. These include different radii for example,(10, 20, 30 and 40 nm), and substrate materials, for example,(polycarbonate and polyester), and two Au surfaces. The inside diameterof the nanochannels may be varied by the plating time, which have beencharacterized for precise nanochannel dimensions. Au nanochannels and Aunanochannels with an adsorbed propanethiol monolayer are preferred.Chloride ions readily adsorb on gold surfaces, thus, in 4.0 M KClreference solutions the Au nanochannels will have a net negative charge.However, the nanochannels pretreated with propanethiol have an inert,uncharged monolayer that prevents chloride ions from adsorbing.

[0179] Alternatively, addition of a propanthiol monolayer isaccomplished by immersing the array into an ethanol solution containingthe thiol. This small thiol molecule does not appreciably change thenanochannel inside diameter when the diameter >˜5 nm. For this reason,there is no need to redetermine the nanochannel inside diameter afterchemisorption of the thiol. In addition, the propanethiol-modified Aunanochannels remain water “wetable” after addition of the thiol. SeeNishizawa, M,; Menon, V. P.; Martin, C. R. Science 1995, 268, 700.

[0180] The various articles of the scientific and/or medical literature,and the U.S. and international and/or foreign patents and patentapplications cited herein are hereby incorporated by reference to theextent permitted by law. To the extent that each is incorporated byreference herein, each constitutes a part of the disclosure of thisspecification. Furthermore, specific embodiments, working examples, andprophetic examples of the invention have been described in detail toillustrate the broad applicability and principles underlying theinvention, such as the use of microfluidic flowing liquid junction aspart of a reference electrode or as part of a combination electrode, andthe various methods of manufacturing and/or using the microfluidicflowing liquid junction, or of manufacturing and/or using a referenceelectrode or a combination electrode comprising a microfluidic flowingliquid junction. Notwithstanding these specific embodiments, workingexamples, and prophetic examples, it will be understood by those ofskill in the art that the invention may be embodied otherwise withoutdeparting from such broad applicability and principles.

What is claimed is:
 1. A flowing junction reference electrodecomprising: a reference electrolyte solution having a viscosity η and apressure P_(E); a sample solution having a pressure P_(S), wherein thedifference between P_(E) and P_(S) is a pressure differential ΔP; aliquid junction member having N discrete nanochannels, the nanochannelshaving diameters D and lengths L; wherein the junction member issituated between the electrolyte solution and the sample solution, andwherein ΔP, D, η, and L are selected such that$\frac{D^{2}\Delta \quad P}{32\quad \eta \quad L}$

 is greater than about 0.1 centimeter per second.
 2. The electrode ofclaim 1, wherein N is less than approximately 100,000 and greater thanapproximately
 10. 3. The electrode of claim 2, wherein N is less thanapproximately 50,000.
 4. The electrode of claim 2, wherein N is lessthan approximately 10,000.
 5. The electrode of claim 2, wherein N isless than approximately 1,000.
 6. The electrode of claim 2, wherein N isgreater than approximately
 100. 7. The electrode of claim 1, wherein adiameter D_(i) of any one nanochannel is substantially equal to adiameter D_(j) of any other nanochannel.
 8. The electrode of claim 1,wherein D is greater than approximately 1 nanometer and less thanapproximately 900 nanometers.
 9. The electrode of claim 1, wherein D isgreater than approximately 10 nanometers and less than approximately 500nanometers.
 10. The electrode of claim 1, wherein the nanochannels arecoated.
 11. The electrode of claim 1, wherein the junction member ismade of a polymer.
 12. The electrode of claim 11, wherein the polymer isselected from the group consisting of polycarbonate and polyimide. 13.The electrode of claim 1, wherein the junction member is made ofsilicon, glass, or ceramic.
 14. The electrode of claim 1, furthercomprising means for maintaining positive linear flow of the electrolytesolution through the nanochannels and into the sample solution.
 15. Theelectrode of claim 14, wherein the means for maintaining positive linearflow of electrolyte flow is selected from the group consisting apressurized collapsible bladder, an electro-osmotic pump, a mechanicalpump, a piezo-electric pump, and a electro-hydrodynamic pump.
 16. Aflowing junction reference electrode comprising: a liquid junctionmember having N discrete nanochannels, each nanochannel having adiameter D and a length L; a reference electrolyte solution passingthrough the member, and having a viscosity η; wherein the member and theelectrolyte are configured such that$\frac{D^{2}\Delta \quad P}{32\quad \eta \quad L}$

 is greater than about 0.1 centimeter per second, wherein ΔP is thedifference between the pressure of the electrolyte as it enters themember and the pressure of the electrolyte as it exits the member.
 17. Acombination electrode comprising the flowing junction referenceelectrode of claim 16 and a sensing electrode.
 18. The combinationelectrode of claim 17, wherein the sensing electrode is selected fromthe group consisting of pH electrodes, other ion-selective electrodes,and redox electrodes.
 19. A flowing junction reference electrodecomprising: a reference electrolyte solution flowing through a liquidjunction member and into a sample solution at a linear flow rate,wherein: the electrolyte solution has a viscosity η and a pressureP_(E); the sample solution has a pressure P_(S) such that P_(E) andP_(S) defining a pressure differential ΔP; the member is situatedbetween the electrolyte solution and the sample solution; the member hasN discrete nanochannels, each nanochannel having a diameter D and alength L; and wherein ΔP, D, η, and L are selected such that the linearflow rate of the electrolyte solution through the nanochannels and intothe sample solution is greater than about 0.1 centimeter per second. 20.The electrode of claim 19, wherein N is greater than approximately 10and less than approximately 100,000.
 21. The electrode of claim 19,wherein N is greater than approximately 10 and less than approximately10,000.
 22. The electrode of claim 19, wherein N is greater thanapproximately 10 and less than approximately 1,000.
 23. The electrode ofclaim 19, wherein N is greater than approximately 10 and less thanapproximately
 800. 24. The electrode of claim 19, wherein N is greaterthan approximately 10 and less than approximately
 400. 25. The electrodeof claim 19, wherein N is greater than approximately 10 and less thanapproximately
 200. 26. The electrode of claim 19, wherein N is greaterthan approximately 10 and less than approximately
 100. 27. The electrodeof claim 19, wherein N is greater than approximately 100 and less thanapproximately 10,000.
 28. The electrode of claim 20, wherein D isgreater than approximately 1 nanometer and less than approximately 900nanometers.
 29. The electrode of claim 20, wherein D is greater thanapproximately 10 nanometers and less than approximately 500 nanometers.30. The electrode of claim 20, wherein D is greater than approximately40 nanometers and less than approximately 250 nanometers.
 31. Theelectrode of claim 20, wherein L is greater than approximately 500micrometers.
 32. The electrode of claim 20, wherein L is greater thanapproximately 0.5 micrometer and less than approximately 300micrometers.
 33. The electrode of claim 20, wherein L is greater thanapproximately 6 micrometers and less than approximately 200 micrometers.34. A flowing junction reference electrode comprising: a liquid junctionmember situated between a pressurized reference electrolyte solution anda sample solution, the junction member having N discrete nanochannels,each nanochannel having a diameter approximately D; wherein N and D aresuch that (i) the pressurized reference electrolyte solution flowsthrough the nanochannels and into the sample solution at a linearvelocity v greater than about 0.1 centimeter per second, and (ii) avolumetric flow q from the electrolyte solution into the sample solutionis less than about 60 micro-liter per hour.
 35. The reference electrodeof claim 34, wherein q is less than approximately 10 microliters perhour.
 36. The reference electrode of claim 34, wherein q is less thanapproximately 1 microliters per hour.
 37. The reference electrode ofclaim 34, wherein v is greater than approximately 0.4 centimeter persecond.
 38. The reference electrode of claim 34, wherein v is greaterthan approximately 4.0 centimeters per second.
 39. The referenceelectrode of claim 34, wherein v is greater than approximately 11.0centimeters per second.